The process, rate and extent of phosphorylation of cytoskeletal proteins are of fundamental importance in normal neuronal function and are likely candidates for pathological dysfunction in certain neurodegenerative disorders, especially Alzheimer's disease. The first goals of this proposed investigation are: 1) to identify the sites on the heat-stable microtubule-associated proteins (MAPs), MAP-2 and tau, that are phosphorylated in vivo, 2) to determine the in vivo rates at which the phosphates turnover on these MAPs, and 3) access the physiological relevance of certain phosphate-transferring enzymes by comparing the sites on MAP-2 and tau that are phosphorylated in vivo with those sites acted upon in vitro by catalytic unit of cyclic AMP-dependent protein kinase, insulin receptor kinase and phosphatase 2B (calcineurin). The importance of this study lies in the more complete delineation of the features of in vivo phosphorylation and is accomplished by using a unique combination of methods recently applied in this laboratory. These techniques include: radiolabeling of phosphoproteins in vivo by intracerebroventricular (icv) administration of 32Pi, focused-beam microwave irradiation (a procedure that rapidly inactivates brain enzymes in situ) to sacrifice animals and isolation of the MAPs by immunoprecipitation with monoclonal antibodies. Subsequent to characterization of the in vivo phosphorylation of MAP-2 and tau, the relationship between specific phosphorylation states of these MAPs and their function(s) will be determined. This objective will be approached by measuring the ability of MAP-2 and tau in defined states of phosphorylation to bind to microtubules and to promote microtubule assembly. The results obtained will provide crucial information about the normal in vivo phosphorylation of MAP-2 and tau and help clarify the role of aberrant cytoskeletal protein phosphorylation in pathological conditions.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29NS027538-05
Application #
3477794
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1989-09-25
Project End
1994-08-31
Budget Start
1993-09-01
Budget End
1994-08-31
Support Year
5
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Alabama Birmingham
Department
Type
Schools of Medicine
DUNS #
004514360
City
Birmingham
State
AL
Country
United States
Zip Code
35294